quareticctivity of Solidago canadensiscultivated ingypt and etermination of theostioactiveraction

Despite the traditional use of Solidago canadensis L. (Asteraceae) as a diuretic drug, there is a scarcity in scientific data concerning the activity of its different extracts and fractions as well as the class of constituents responsible for this diuretic action. A comparative study was carried out for the diuretic activities of the different standardized extracts and fractions of the flowering aerial parts of S. canadensis, as well as isolation of compounds from the most biologically active fraction. The ethanol extract and its ethyl acetate fraction (EA) showed the highest aquaretic activities (91 and 58% at a dose of 400 mg/Kg b.wt., respectively) compared to 100% of furosemide at 20 mg/Kg b.wt.. Their activities were higher than Cystinol® and spironolactone reference standards (74% and 59% of furosemide, respectively). EA showed the highest total phenolic and flavonoid contents among the tested fractions of the ethanol and aqueous extracts (9.38 ± 0.004 g GAE and 39.75 ± 0.005 g RE/100 g dried extract, respectively). Eight flavonoids, 2 phenolic acids and 1 nucleoside were isolated from EA. This is the first report of a comparative study between the aquaretic activities of the different extracts, fractions and essential oil of S. canadensis, as well as isolation of thyimidine (1), isorhamnetin-3-O-β-ᴅ-glucopyranoside (2), kaempferol-3-O-(6”-O-acetyl)-β-ᴅ-glucopyranoside (4), quercetin-3-O-(6”-O-acetyl)-β-ᴅ-glucopyranoside (5), and kaempferol-3-O-β-ᴅ-apiofuranoside (7) from genus Solidago.


Introduction
Phytopharmaceuticals were successfully used in the therapy of the urinary tract with parallel administration of synthetic drugs especially those used as diuretics (1). Diuretics are commonly defined as drugs that promote the rate of urine flow by the kidneys (2). The commonly used synthetic diuretics (viz thiazides and furosemide) have been associated with many side effects such as disturbances of electrolytes, acid-base and water balance, changes in uricacid, carbohydrate and lipid metabolism, and drug interactions (3). Therefore, herbal diuretics could be considered as a better therapeutic option, having relatively safer and milder actions, compared to synthetic diuretics which cause several adverse effects due to their strong saluretic actions (4). Numerous herbs were traditionally considered as diuretics. Among those herbs are members of genus Solidago belonging to family Asteraceae (5).
In the past few years, the extract of the flowering aerial parts of Solidago virgaurea L. was launched in the Egyptian market under the trade name of Cystinol® at a dose of 400 mg. It is used for the treatment of urolithiasis by promoting the excretion of water more than the electrolytes and increasing renal blood flow. This facilitates the washout of bacteria from the urinary tract, prevents crystal formation, and hence kidney stones (46).
Having Cystinol® in the Egyptian market, it was found interesting to investigate the diuretic activity of the essential oil and crude extracts of the flowering aerial parts of S. canadensis cultivated in Egypt using different solvents for extraction (70% ethanol and water). A biologically guided fractionation of the crude extracts was carried out to determine the most active fraction and its content of active constituents.

Plant material
Samples of Solidago canadensis L. were collected during the years 2010-2013 from El-Mansoureya, Giza, Egypt. The plant was kindly authenticated by the temperate regional team of the Royal Botanical Gardens, Kew., London, U.K. Voucher specimen of the plant (24.02.2013) was deposited at the herbarium of the Pharmacognosy Department, Faculty of Pharmacy, Cairo University.

Plant extraction A-Ethanolic extract
The air-dried powdered flowering aerial parts of S. canadensis (1 kg) was exhaustively extracted with 70% ethanol by cold maceration to give 230 g extract. An aliquot of the dry residue (200 g) was subjected to liquid-liquid fractionation with n-hexane, methylene chloride, ethyl acetate and n-butanol saturated with water and concentrated to give 10.4, 6.8, 17.2 and 22 g of dried fractions, respectively. The remaining water of the ethanol extract was lyophilized and weighed (122 g).

B-Aqueous extract
The air-dried powdered flowering aerial parts (1 kg) were macerated in boiling distilled water for 20 min. The aqueous extract was lyophilized to give 206 g residue. An aliquot of the lyophilized residue (200 g) was extracted with Aquaretic activity of Solidago canadensis L. methylene chloride, ethyl acetate and n-butanol saturated with water to give 12, 18, and 26 g of the dried fractions, respectively. The remaining water left after fractionation was lyophilized and weighed (138 g). The extracts and fractions were stored at 5 °C for both the phytochemical and biological investigations.

C-Preparation of essential oil
The fresh flowering aerial parts (200 g) were subjected to hydrodistillation using a Clavenger′s apparatus according to the procedures described in the Egyptian Pharmacopoeia (47). The obtained essential oil sample wasdried over anhydrous sodium sulphate. The hydrodistilled oil was saved in a refrigerator (4 ºC) in a tightly sealed container.

Chemicals and equipment
Cystinol® was purchased from Atos Pharma (Cairo, Egypt). Furosemide and spironolactonewere obtained from Sigma-Aldrich (Darmstadt, Germany) and used as reference diuretic drugs.

Evaluation of the pharmacological activity Animals
Adult male albino rats of Wistar strain (120-150 g), obtained from the animal house colony at the National Research Center (Dokki, Giza, Egypt), were utilized for determination of the LD 50 and assessment of the diuretic activity. They were housed in steel cages at standardized conditions of temperature and humidity and fed with standard pellets and water ad libitum. All experimental procedures were conducted in accordance with the internally accepted principles for laboratory animal use and care, and were approved by the Ethics Committee No. MP (4) in accordance with recommendations for the proper care and use of laboratory animals (NIH Publication No. 85-23;revised 1985).

Determination of median lethal dose (LD 50 )
The LD 50 of both the ethanol and aqueous extracts was determined following Karber′s procedure (1931) (52). Five groups, each of six rats, received both plant extracts separately in doses ranging from 1 to 4 g/kg b.wt. The LD 50 of the tested extracts was calculated according to the following formula: Where: Dm = The largest dose that killed all animals. z = Mean of dead animals between 2 successive groups. d = The constant factor between 2 successive doses. n = Number of animals in each group. Σ = The sum of (z × d).

Evaluation of the diuretic activity
The 70% ethanol and aqueous extracts, their fractions, and the essential oil of the flowering aerial parts were tested for their diuretic activities as well as their effect on the excretion of potassium and sodium in urine according to the method of Lipschitz et al. 1943 (53). Three diuretic drugs namely furosemide (20 mg/kg b.wt.), spironolactone (25 mg/kg b.wt.) and Cystinol® (400 mg/kg/b.wt.) were used as reference standards. Oral doses of 200 and 400 mg/kg b.wt.of the tested Solidago extracts and fractions were selected for the study, based on the marketed dose of Cystinol®. The rats were fasted and deprived of water for 18 h before the experiment. They were divided into 28 groups of six animals each. The rats of each group were subjected to the specified treatment and the control group received 1 mL 0.9% NaCl/100 g b.wt. The following parameters were estimated:

Urine output
Immediately after the treatment, the animals were individually placed in metabolic cages specially designed to separate urine and faeces. During this period, neither food nor water was made available to the animals. The room temperature was maintained at 27-29 °C. The urine was collected in measuring cylinders up to 24 h after treatment for all control and treated groups. Urine volume was expressed as mL/kg (54).

Estimation of electrolytes
The electrolyte (Na + , K + ) content was estimated in the urine using the commercially available kit (Biodiagnostic Co., Giza, Egypt).

Statistical analysis
The data obtained were presented as mean ± standard error (SE) and statistically analyzed using ANOVA followed by LSD post-hoc test. The values were determined to be significant when p-value was less than 0.05 (p < 0.05).

Isolation of the components of the ethylacetate fraction of the 70% ethanolextract (EA)
EA (16 g) was chromatographed on a Diaion column (35 cm L × 3.5 cm D). Gradient elution with water/methanol mixtures was adopted. Fractions, 200 mL each, were collected and monitored by TLC using solvent system S 2 . Similar fractions were pooled together and the solvents were separately evaporated under reduced pressure yielding three major fractions (I-III). Fraction I (0.8 g, eluted with 25% methanol in water) was rechromatographed over a silica gel 60 column (30 × 1 cm) eluted with methylene chloride: methanol (95:5 v/v) yielding compound 1 (30 mg). Fraction II (6.5 g, eluted with 50% methanol in water) was rechromatographed over a sephadex LH 20 column (30 × 3 cm) using methylene chloride: methanol (1:1 v/v) as eluent to give three fractions (IIa, IIb and IIc). Upon evaporation of fractions IIa and IIb, two pure compounds 6 m 1 to 4 g/kg b.wt. The LD50 of the tested extracts was calculated according ormula: animals between 2 successive groups.
actor between 2 successive doses. imals in each group.
diuretic activity nol and aqueous extracts, their fractions, and the essential oil of the arts were tested for their diuretic activities as well as their effect on the sium and sodium in urine according to the method of Lipschitz et al. 1943 tic drugs namely furosemide (20 mg/kg b.wt.), spironolactone (25 mg/kg ol® (400 mg/kg/b.wt.) were used as reference standards. Oral doses of 200 wt.of the tested Solidago extracts and fractions were selected for the study, eted dose of Cystinol®. The rats were fasted and deprived of water for 18 h ment. They were divided into 28 groups of six animals each. The rats of subjected to the specified treatment and the control group received 1 mL b.wt. The following parameters were estimated: r the treatment, the animals were individually placed in metabolic cages d to separate urine and faeces. During this period, neither food nor water le to the animals. The room temperature was maintained at 27-29 °C. The ed in measuring cylinders up to 24h after treatment for all control and ine volume was expressed as mL/kg (54).

Urinary excretion of test group
Urinary excretion of control group (55, 56).

Diuretic action of extract
Diuretic action of standard x 100 (55). 6 each group were subjected to the specified treatment and the c 0.9% NaCl/100 g b.wt. The following parameters were estimated

Urine output
Immediately after the treatment, the animals were individuall specially designed to separate urine and faeces. During this pe was made available to the animals. The room temperature was urine was collected in measuring cylinders up to 24h after t treated groups. Urine volume was expressed as mL/kg (54).

Diuretic action:
Urinary excretion of test group Urinary excretion of control group (55, 56).

Diuretic activity:
Diuretic action of extract Diuretic action of standard x 100 (55). 6 0.9% NaCl/100 g b.wt. The following parameters were estimated:

Urine output
Immediately after the treatment, the animals were individually p specially designed to separate urine and faeces. During this perio was made available to the animals. The room temperature was ma urine was collected in measuring cylinders up to 24h after trea treated groups. Urine volume was expressed as mL/kg (54).

Diuretic action:
Urinary excretion of test group Urinary excretion of control group (55, 56).

Diuretic activity:
Diuretic action of extract Diuretic action of standard x 100 (55). 6 0.9% NaCl/100 g b.wt. The following parameters were estimate

Urine output
Immediately after the treatment, the animals were individual specially designed to separate urine and faeces. During this p was made available to the animals. The room temperature was urine was collected in measuring cylinders up to 24h after treated groups. Urine volume was expressed as mL/kg (54).

Diuretic action:
Urinary excretion of test group Urinary excretion of control group (55, 56).

Diuretic activity:
Diuretic action of extract Diuretic action of standard x 100 (55). 6 each group were subjected to the specified treatment and the control g 0.9% NaCl/100 g b.wt. The following parameters were estimated:

Urine output
Immediately after the treatment, the animals were individually placed specially designed to separate urine and faeces. During this period, ne was made available to the animals. The room temperature was maintai urine was collected in measuring cylinders up to 24h after treatmen treated groups. Urine volume was expressed as mL/kg (54). x 100 (55). 6 before the experiment. They were divided into 28 groups of each group were subjected to the specified treatment and the 0.9% NaCl/100 g b.wt. The following parameters were estimate

Urine output
Immediately after the treatment, the animals were individual specially designed to separate urine and faeces. During this p was made available to the animals. The room temperature was urine was collected in measuring cylinders up to 24h after treated groups. Urine volume was expressed as mL/kg (54). 6 based on the marketed dose of Cystinol®. The rats were fasted and de before the experiment. They were divided into 28 groups of six an each group were subjected to the specified treatment and the contro 0.9% NaCl/100 g b.wt. The following parameters were estimated:

Urine output
Immediately after the treatment, the animals were individually pla specially designed to separate urine and faeces. During this period, was made available to the animals. The room temperature was maint urine was collected in measuring cylinders up to 24h after treatm treated groups. Urine volume was expressed as mL/kg (54).

Statistical analysis
The data obtained were presented as mean ± stand using ANOVA followed by LSD post-hoc test. The when p-value was less than 0.05 (p < 0.05).

Determination of median lethal dose
Both the ethanol and aqueous extracts were safe and non-toxic under the present   (57).

Effect on urine volume
The reference diuretic drugs furosemide, spironolactone, and Cystinol® significantly increased the urine output when compared to the control group (Table 5). The diuretic activities of the tested samples were calculated relative to furosemide, as it proved to be the most potent diuretic reference drug. All the tested samples showed a significant increase in the volume of urine output except the n-butanol and the remaining water fractions of the aqueous extract (Table 5). The 70% ethanolextract of the aerial parts (400 mg/ kg/b.wt.) exhibited the highest diuretic activity of all tested samples comparable to that of furosemide (91% of furosemide activity), and higher than spironolactone and Cystinol® which exhibited 59% and 74% of furosemide activity, respectively ( Table 5). The aqueous extract (400 mg/kg/b.wt.) exhibited lower diuretic activity than that of the 70% ethanol extract and all the used reference diuretics (46% of furosemide). Whereas, the ethyl acetate fraction of the 70% ethanol extract (EA) (400 mg/kg/b.wt.) showed the highest diuretic activity amongst all fractions representing 58% of furosemide activity and compared to spironolactone (potassium sparing diuretic). However, it was less potent than the parent 70% ethanol extract at the same dose level. The essential oil (400 mg/kg/b.wt.) showed poor    diuretic activity (31% of furosemide activity).

Effect on urinary electrolyte excretion
Furosemide significantly increased the excretion of urinary electrolytes. Spironolactone (potassium sparing diuretic) increased the excretion of sodium iononly, while Cystinol® did not affect the excretion of sodium and potassium in urine (aquaretic). Administration of all the tested extracts, fractions and the essential oil of the aerial parts at both dose levels (200 and 400 mg/kg b.wt.) did not affect the urinary electrolyte excretion (Table 5). The Na + / K + excretion ratio was uniform (1.27 to 1.36) in all the tested plant samples.

Spectrophotometric estimation of the total phenolic, flavonoid and saponin contents
The 70% ethanol extract of the total aerial parts showed higher total phenolic and flavonoid contents than their aqueous extract while the saponins were more concentrated in the aqueous Table 5. The diuretic action, diuretic activity, Na + , K + levels and Na + /K + ratios of the essential oil, extracts and fractions of the aerial parts of Solidago canadensis L.  extract. The most active EA showed the highest total phenolic and flavonoid contents amongst all the tested fractions of both extracts, whereas the n-butanol fraction of the aqueous extract was the highest in the saponin content ( Table 6).

Discussion
Preliminary phytochemical screening of the air-dried flowering aerial parts of S. canadensis deduced that the main constituents of the plant were essential oils, free and combined flavonoids, and saponins. Guided by the available literature and the results of the phytochemical screening, it was found interesting to study the main active constituents of the plant and the solvent of choice for the extraction of each class of constituents knowing that the essential oil of the plant was previously investigated by our group (35). Higher yield of total phenolic compounds and flavonoids was achieved by extraction with 70% ethanol rather than water. This was in accordance with the previous reports of Apati et al. 2002 (39). EA showed the highest diuretic activity as well as the highest total phenolic and flavonoid contents relative to the other fractions, this made it the most proper candidate for further phytochemical investigations. Optimum extraction of saponins was achieved by water rather than 70% ethanol. The saponins were concentrated in the n-butanolfraction of the 12`3`4561`2`3`4```5`6`1``2``3``5``6```
Previous reports claimed that the flavonoids and saponins of the different Solidago species were responsible for the diuretic activity of the genus (7, 43 and 44). Others attributed the activity to their content of flavonoids and phenolics (6, 9 and 68). In our study, the highest diuretic activity of all tested samples was exhibited by the 70% ethanol extract. This high potency was presumably related to its high content of phenolics and flavonoids. This was further confirmed by the high diuretic activity of EA which possessed the highest phenolic and flavonoid contents among other fractions, the inactivity of the saponin rich n-butanol fraction of the aqueous extract, as well as the poor activity of the essential oil. This was in accordance with Apati et al. 2003 who stated that the flavonoids, especially quercetin and its derivatives showed a potential to inhibit the neutral endopeptidase enzyme, which is responsible for the interaction of the atrial natriuretic peptide through the excretion of the sodium ions (68). Moreover, the flavonoid fractions of some previously studied Solidago species showed diuretic activities, and the diuretic actions of several plant species were related to their flavonoid content (9, 69-71). The fractions of the 70% ethanol and aqueous extracts proved to be less active than the parent extracts, except for the ethyl acetate fraction of the aqueous extract, suggesting the existence of additive and/or synergistic effects in the parent extracts. The 70% ethanol extract of the flowering aerial parts of S. canadensis showed more powerful diuretic activity than the aquaretic drug Cystinol® (91% and 74% of furosemide activity, respectively) at the same dose level (400 mg/kg b.wt.) with a similar aquaretic property. It also showed a much higher diuretic activity when compared to the potassium sparing diuretic spironolactone (91% and 59% of furosemide activity, respectively) but without promoting the loss of sodium in urine. The tested 70% ethanol extract showed a comparable diuretic activity to the loop diuretic furosemide but without enhancing the loss of the electrolytes in urine (Na + and K + ). The increase in urine volume without loss of electrolytes showed that the tested samples were aquaretics, similar to the reference drug Cystinol®.
The spectral data of compound 1 showed that the protons were assigned to the pyrimidine base thymine and deoxyribose sugar. The imino proton of the thymine base appeared at δ 11.27 ppm. The olefinic proton H-6 appeared at δ 7.70 ppm. The 3 protons of the methyl group at C-5 appeared as a singlet integrated as 3H at δ 1.78 ppm. The H-2′, H-3′, H-4′, and H-5′ protons of the deoxyribose sugar appeared at the region between 2.03 -4.22 ppm. The anomeric proton of the deoxyribose sugar appeared as a triplet at δ 6.15 ppm. The 13 C-NMR spectrum of compound 1 revealed the presence of ten carbon atoms in the molecule. The 13 C chemical shifts of a carbon at δ 12.72 indicated the presence of amethyl group attached to C-5. The assignment of the carbons of the pyrimidine base was determined using HMBC spectrum. Compound1 was identified as thymidine. Compound 7 showed the signals characteristic for a kaempferol nucleus and additional signals for a sugar moiety. The spectrum showed anomeric proton at 5.01 ppm as a doublet with J= 3.7 Hz characteristic for O-β-ᴅ-apiofuranose structure (63, 64, 72 and 73). 13 C-NMR spectrum of compound 7 showed 18 carbon signals assigned to 20 carbons, 13 of which assigned to kaempferol (15 carbons) and 5 for apiose sugar. Signal of C-3 at 133.10 δ ppm was shifted upfield by 2 ppm relative to kaempferol aglycone (74). This confirmed the 3-glycosylation of kaempferol. Compound 7 was identified as kaempferol-3-O-β-ᴅ-apiofuranoside. 1 H-NMR spectrum of compound 9 showed the characteristic signals for a caffeic acid molecule. Also the protons of a quinic acid moiety could be observed with a doublet at δ 1.73 (J = 12.9 Hz) assigned to H-6 ax, a doublet of doublet at δ 1.90 ppm (J = 13.8, 10.8 Hz) assigned to H-6 eq and a multiplet at 2.08 ppm integrated as two protons and assigned to H-2 ax and H-2 eq . A broad singlet at δ 3.82 and a doublet at δ 3.87 ppm (J = 9 Hz) assigned to H-4 and H-5. The downfield shift of H-3 which appeared at δ 5.01 ppm indicated the acylation of the quinic acid by the caffeic acid at the OH on C-3 (75). The assignement of the protons of the quinic acid moiety was determined using 1 H-1 H COSY. Compound 9 was identified as neochlorogenic acid.